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Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October-January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Type of study / information:
In this study the potential to modulate the steroidogenic pathway, beginning with the sequence of reactions occurring after the gonadotropin hormone receptors through the production of testosterone and estradiol, was investigated using the human H295R cell line.
Test guideline
Qualifier:
according to
Guideline:
other: OECD 456
Version / remarks:
adopted 28 July 2011
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
White solid
CAS nr 288-88-0
Storage at room temperature

Results and discussion

Any other information on results incl. tables

In the solubility test, the test item dissolved at a concentration of 1 M in DMSO. Thousand-fold dilution of this stock solution in exposure medium (final concentration: 1 mM) resulted in a clear solution. Therefore 1 mM was used as the highest test item concentration in the steroidogenesis assay experiments.

The results of the chemical hormone interference showed that compared to the basal hormone production, the interference of 1,2,4-Triazole with estradiol and testosterone hormone measurements was 18% and 7%, respectively. Since the interference observed for testosterone and estradiol hormone measurement was below 20%, the interference of the test item with the hormone assays was considered to be not significant.

Medium samples spiked with 500 pg/mL and 2500 pg/mL testosterone or 50 pg/mL and 250 pg/mL estradiol were prepared and analyzed in the EIA to determine the recovery. The results of the hormone recovery tests for estradiol and testosterone showed that the recovery of the estradiol spikes was 80-106% and the recovery of the testosterone spikes was 84-98%. Since the measured hormone concentrations did not deviate more than 30% of the nominal concentrations, the EIA assays were considered to be suitable for estradiol and testosterone analysis.

Four steroidogenesis exposure experiments were performed. The data of experiment 3a were rejected since the wrong exposure concentrations were used. This exposure experiment was repeated with the correct exposure concentrations (experiment 3b). The data of experiment 1, 2 and 3b are discussed in this results section.

After each exposure experiment the viability of the H295R cells was determined using the MTT test. The test item did not affect cell viability at any of the concentrations tested.

To evaluate the performance of the assay a quality control (QC) plate was included in each of the three steroidogenesis assay experiments.  A comparison of the results obtained for the QC and test item plates with the acceptability criteria for estradiol and testosterone is presented in the tables below.

Acceptability Criteria for Estradiol obtained in Experiment 1, 2 and 3b

 

Comparison between

Estradiol

Performance criteria

Experiment 1

Experiment 2

Experiment 3b

QC plate

LOQ

NA

NA

6.9 pg/mL

6.2 pg/mL

6.4 pg/mL

Basal production of hormone in the SCs

Absolute concentration

NA

117 pg/mL

139 pg/mL

180 pg/mL

Fold-greater than LOQ

>=2.5-fold

17-fold

22-fold

28-fold

Induction
(10 µM forskolin)

Fold-change

compared to SC

>= 7.5-fold

7.5-fold

14-fold

15-fold

Inhibition
(1 µM prochloraz)

Fold-change

compared to SC

= <0.5-fold

0.3-fold

0.3-fold

0.2-fold

Test item plate

LOQ

NA

NA

9.8 pg/mL

5.9 pg/mL

7.6 pg/mL

Basal production of hormone in the SCs

Absolute concentration

NA

144 pg/mL

167 pg/mL

194 pg/mL

Fold-greater than LOQ

>= 2.5-fold

15-fold

28-fold

26-fold

Within plate CV for SCs (replicate wells)

Absolute concentrations

= <30%

8%

11%

12%

Hormone measurement system: Sensitivity

Detectable fold-change relative to SC

>= 2.5-fold

15-fold

28-fold

26-fold

Hormone measurement system: Replicate measure CV for SCs

Absolute concentrations

= <25%

= <20%

= <22%

=< 25%

Acceptability Criteria for Testosterone obtained in Experiment 1, 2 and 3b

 

Comparison between

Testosterone

Performance criteria

Experiment 1

Experiment 2

Experiment 3b

QC plate

LOQ

NA

NA

6.3 pg/mL

6.3 pg/mL

7.2 pg/mL

Basal production of hormone in the SCs

Absolute concentration

NA

2760 pg/mL

3122 pg/mL

2778 pg/mL

Fold-greater than LOQ

>=5-fold

438-fold

496-fold

386-fold

Induction

(10 µM forskolin)

Fold-change

compared to SC

>= 1.5-fold

2.3-fold

2.2-fold

2.6-fold

Inhibition

(1 µM prochloraz)

Fold-change

compared to SC

= <0.5-fold

0.2-fold

0.1-fold

0.1-fold

Test item plate

LOQ

NA

NA

8.8 pg/mL

6.1 pg/mL

9.2 pg/mL

Basal production of hormone in the SCs

Absolute concentration

NA

2318 pg/mL

2679 pg/mL

2877 pg/mL

Fold-greater than LOQ

=>5-fold

263-fold

443-fold

313-fold

Within plate CV for SCs (replicate wells)

Absolute concentrations

= <30%

7%

1%

7%

Hormone measurement system: Sensitivity

Detectable fold-change relative to SC

=> 5-fold

263-fold

443-fold

313-fold

Hormone measurement system: Replicate measure CV for SCs

Absolute concentrations

=<25%

=< 21%

= <14%

= <17%

Based on the results obtained for the QC plates it was concluded that in all three experiments the acceptability criteria were met for both estradiol and testosterone.

Results Steroidogenesis Assay Experiment 1

In steroidogenesis assay experiment 1, the tested concentrations were 0.001, 0.01, 0.1, 1, 10, 100 and 10000 µM of 1,2,4-Triazole. The mean basal estradiol production in the DMSO solvent control wells was 144 pg/mL. The estradiol concentrations in medium from H295R cells exposed to the test item varied from 149 pg/mL to 165 pg/mL. The relative change of test item exposed wells compared to the solvent treated control wells varied from 1.04 to 1.15. Statistical analysis showed that 1,2,4-Triazole had no significant effect (p =0.05) on estradiol release of H295R cells.

The mean basal testosterone production in the DMSO solvent control wells was 2318 pg/mL. The testosterone concentrations in the medium from the H295R cells exposed to the test item varied from 1585 pg/mL to 2993 pg/mL. The relative change of test item exposed wells to solvent treated control wells varied from 0.68 to 1.29. Statistical analysis showed that 1,2,4-Triazole significantly (p =0.05) decreased testosterone release in H295R cells at a concentration of 0.1 µM and significantly increased testosterone release at a concentration of 100 µM.

Since the effect observed for testosterone was not dose-related, i.e. no statistically significant effects at two (or more) adjacent concentrations, the results of the first steroidogenesis experiment were considered to be equivocal.

Results Steroidogenesis Assay Experiment 2

Based on the results obtained in experiment 1, ½-log concentrations were included in the second experiment bracketing the concentrations that elicited an effect (0.1 µM and 100 µM). This was designated to better characterize any potential effect of the test item on testosterone release. The following concentrations were tested in experiment 2: 0.03 µM, 0.1 µM, 0.3 µM, 30 µM, 100 µM, 300µM and 1000µM.

In steroidogenesis assay experiment 2, the mean basal estradiol production in the DMSO solvent control wells was 167 pg/mL. The estradiol concentrations in medium from H295R cells exposed to the test item varied from 127 pg/mL to 188 pg/mL. The relative change of test item exposed wells compared to the solvent treated control wells varied from 0.76 to 1.12. Statistical analysis showed that 1,2,4-Triazole had no statistical significant effect (p =0.05) on estradiol release of H295R cells.

The mean basal testosterone production in the DMSO solvent control wells was 2679 pg/ml. The testosterone concentrations in the medium from the H295R cells exposed to the test item varied from 2120 pg/mL to 2838 pg/mL. The relative change of test item exposed wells to solvent treated control wells varied from 0.79 to 1.06. Statistical analysis showed that 1,2,4-Triazole significantly (p =0.05) decreased testosterone release in H295R cells at a concentration of 0.03 µM.

Since exposure of 0.1 µM and 100 µM of 1,2,4-Triazole had no effect on testosterone release in experiment 2, the results could not confirm the effects observed in the first experiment. The significant decrease in testosterone observed at 0.03 µM was considered to be assay variation. Therefore, the results obtained in steroidogenesis experiment 2 were considered to be negative. This was supported by the results obtained in experiment 3a where no significant effect on estradiol and testosterone release was observed when using the same exposure concentrations as in experiment 2.

Results Steroidogenesis Assay Experiment 3b

Since the second experiment did not confirm the results of the first experiment, according to OECD guideline 456 recommendation, the third experiment was conducted using the test concentrations of the first experiment.

In steroidogenesis assay experiment 3b, the mean basal estradiol production in the DMSO solvent control wells was 194 pg/mL. The estradiol concentrations in medium from H295R cells exposed to the test item varied from 177 pg/mL to 201 pg/mL. The relative change of test item exposed wells compared to the solvent treated control wells varied from 0.91 to 1.04. Statistical analysis showed that 1,2,4-Triazole had no significant effect (p =0.05) on estradiol release of H295R cells.

The mean basal testosterone production in the DMSO solvent control wells was 2877 pg/ml. The testosterone concentrations in the medium from the H295R cells exposed to the test item varied from 2431 pg/mL to 3205 pg/mL. The relative change of test item exposed wells to solvent treated control wells varied from 0.84 to 1.11. Statistical analysis showed that 1,2,4-Triazole had no significant effect (p =0.05) on testosterone release of H295R cells.

Since no statistical significant effects on estradiol and testosterone release were observed the test item was considered to be negative in steroidogenesis experiment 3b.

The equivocal results in the first experiment could not be confirmed in the two subsequent experiments (both negative), and therefore also the results of the first experiment were consequently considered to be negative. Overall it can be concluded that 1,2,4-Triazole did not alter estradiol and testosterone release in H295R cells and that the No Observed Effect Concentration (NOEC) is 1 mM, the highest concentration tested, and the maximum concentration to be assayed according to OECD Test Guideline 456.

Applicant's summary and conclusion

Conclusions:
In conclusion, all H295R steroidogenesis assay experiments were considered to be valid. The test item 1,2,4-Triazole did not alter estradiol and testosterone release in H295R cells and therefore was considered to be negative in the steroidogenesis assay.
Executive summary:

In this study the potential of 1,2,4-Triazole to modulate the steroidogenic pathway, beginning with the sequence of reactions from cholesterol through the production of testosterone and estradiol, was investigated using the human H295R cell line according to OECD Test Guideline456.

Three valid steroidogenesis assay experiments (designated 1,2, and 3b) were performed whereby 1,2,4 -Triazole was tested at seven concentrations up to and including1 mM together with the positive control inducer forskolin and positive control inhibitor prochloraz. After the first two experiments (1 and 2), an initial third experiment (designated Experiment 3a) was performed, but the concentrations chosen were incorrect (same concentrations used as in experiment 2 instead of experiment 1). Therefore, a further experiment (designated Experiment 3b) was performed with the correct concentrations.

Dimethylsulfoxide (DMSO) was used as vehicle and the concentration of vehicle in the incubations was kept constant at 0.1% (v:v).

H295R cells were exposed for 48 hours to the vehicle, 1,2,4 -Triazole and positive controls. After exposure, the viability of the cells was determined using the 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The concentration of estradiol and testosterone in the exposure medium was determined using commercial Enzyme Immuno Assays (EIAs).

1,2,4 -Triazole did not affect cell viability up to and including a concentration of 1 mM. No interference of the test item with the estradiol and testosterone EIAs was observed.

The assay acceptability criteria i.e. the basal hormone production, within plate coefficient of variation (CV) for the DMSO solvent controls, hormone measurement sensitivity, induction of hormone production upon exposure of 10 µM forskolin exposure and inhibition of hormone production upon exposure of 1 µM prochloraz were all met. Therefore, all steroidogenesis assay experiments were considered to be valid.

1,2,4 -Triazole had no statistically significant effect on estradiol release in H295R cells in any of the three experiments. In experiment 1, testosterone release was statistically significant decreased at a test item concentration of 0.1 µM and significantly increased at a concentration of 100 µM. Since no dose related effect was observed for testosterone, i.e. no statistically significant effect at two (or more) adjacent concentrations, the results of the first steroidogenesis experiment initially were considered to be equivocal. In experiment 2, testosterone release was only significantly decreased at a concentration of 0.03 µM. In experiment 3b, no significant effect on testosterone release was observed at any of the test item concentrations.

The equivocal results on testosterone release in the first experiment could not be confirmed in the two subsequent experiments, and therefore also the results of the first experiment were considered to be negative. The No Observed Effect Concentration (NOEC) was 1 mM which was the highest concentration tested and the maximum required test concentration to be assayed according to OECD Test Guideline 456.

In conclusion, all H295R steroidogenesis assay experiments were considered to be valid. The test item 1,2,4-Triazole did not alter estradiol and testosterone release in H295R cells and therefore was considered to be negative in the steroidogenesis assay.